| The development of gonad is one of the most important fields in development biology, which is regulated by many genes and is affected by sex hormones and environmental factors. At present, it has been well known that the sex hormones function in the process of gonad development. However, the molecular mechanism that the sex hormones are activated and inactivated is only described in mammals. Molluscs is one of important groups in animal kingdom, datas are insufficient to understand the synthesis and degradation of sex hormones during gonad development and regulation. In this study, the17β-HSD4and17β-HSD14which can inactivate estradiol, androstenediol and5a-androstane-3α,17β-diol were selected as the research object. Two partial expression sequence tags (EST) which were great similarities with17β-HSD4and17β-HSD14of other species were obtained from the transcription group database of Chlamys farreri and both of cDNA full-lengh sequcences were cloned using Rapid Amplification of cDNA End methods (RACE). Furthermore, semi-quantitative and quantitative RT-PCR were employed respectively to detecte their expressions in the major tissues and gonad during the development in C. farreri. The results are as follows.The full-length of17β-HSD4was2217bp in C. farrert consisting of an open reading frame (ORF) of2223bp encoding a deduced peptide of740amino acids which contained4conserved domains of SDR super family and3enzyme structural domains of17β-HSD4. The deduced sequence had identity of over58%with the sequence of vertebrates. Semi-quantative RT-PCR detected the mRNA expressed widely in the testis, ovary, adductor muscle, mantle, gill, hepatopancreas and kidney, especially higher in hepatopancreas and kidney, suggesting17β-HSD4could be synthetized in many tissues of C. farreri. Result of quantitative RT-PCR indicated its expression pattern was different between testis and ovary during the development cycle of gonad, and the expression levels in testis of growing stage and mature stage were obviously higher than that in ovary at the same stage (P<0.05). It was supposed that the gene might be more important in regulating the development and maturity of testis than ovary, and took role in the gonadal development by β-oxidation to regulate the accumulation the lipids.The cDNA full-length of17β-HSD14was2217bp in C.farreri consisting of an ORF of819bp encoding a putative peptide of272amino acids. The amino acids sequece contained4conserved domains of SDR super family, and Lys158-Tyr154-Ser141triplet residue which was corresponding to the ternary compound structure of17β-HSD14/NADP/estradiol, and had identity of over50%with the17β-HSD14of other species. Quantative RT-PCR reveals its expression patterns in testis and ovary at different development stages of gonad were similar to that of17β-HSD4, i.e. the expression in testis at growing stage and mature stage was obviously higher than that in ovary at the same stages (P<0.05), and it was obviously higher in testis of mature stage than that of othe stages. It could be concluded that17β-HSD14might have the activity of hydroxysteroid dehydrogenase in C. farreri gonad, and also played an important role in regulating the level of estradiol.Furthermore,10tags were selected based on differentially expresses profile between the testis and ovary at proliferative stage in C.farreri. And semi-quantitative RT-PCR detected that two were ovary specific expression tags in ovary,4higher expression tags in ovary and1higher expression tag in testis. The primers were designed based on a specific expression tag (>FTYGYVA03G8KDK) in ovary, and its cDNA fragment of1142bp containing22bp continuous poly(A) at3’end is obtained. Interestingly, the sequence was not matched to any homologous sequence in GenBank. Based on its expression only in ovary, while no expression in other tissues of C. farreri, we hypothesized that the gene might be a novel gene taking part in sex differentiation and ovary devepment in C.farreri. |
PDF Full Text Request